CN110646542A

CN110646542A - Quality detection method for salvia miltiorrhiza medicinal material

Info

Publication number: CN110646542A
Application number: CN201910944994.5A
Authority: CN
Inventors: 李玮
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou University of Traditional Chinese Medicine
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2020-01-03

Abstract

The invention relates to a quality detection method of a salvia miltiorrhiza medicinal material, establishes an HPLC fingerprint spectrum determination method of the salvia miltiorrhiza medicinal material, and has the advantages of simple and convenient operation, stability, high precision and good reproducibility; the established common mode of the HPLC fingerprint of the salvia miltiorrhiza medicinal material shows that the similarity of the salvia miltiorrhiza medicinal material and the salvia miltiorrhiza medicinal material of different processing methods is mostly more than 0.900 according to the analysis result of the correlation coefficient, so that the chemical components of the salvia miltiorrhiza medicinal material and the salvia miltiorrhiza medicinal material of different processing methods are relatively stable, and the safety and the effectiveness of the medicinal materials are improved.

Description

Quality detection method for salvia miltiorrhiza medicinal material

Technical Field

The invention relates to the technical field of traditional Chinese medicine quality detection, in particular to a quality detection method for a salvia miltiorrhiza medicinal material.

Background

The Saviae Miltiorrhizae radix is dried root and rhizome of Salvia officinalis Bge (Salvia militaria Bge.) of Labiatae, and is mainly produced in Shandong, Henan, Hebei, Sichuan and Anhui provinces. Guizhou has a salvia miltiorrhiza planting base. The Saviae Miltiorrhizae radix has effects of promoting blood circulation, removing blood stasis, dredging channels, relieving pain, resisting bacteria and diminishing inflammation. Has very obvious effect on blood circulation diseases and cardiovascular diseases, is one of the effective components of the compound red sage root drop pill which is a Chinese patent medicine with large clinical dosage and is only listed in the United states and approved by the FDA in the United states in China.

As for the quality detection method of salvia miltiorrhiza, the team of the invention has carried out a series of researches, and one of the inventors has published a paper (influence of a processing method on cryptotanshinone and tanshinone IIA in salvia miltiorrhiza, Guizhou agricultural science 2014, 42(9): 190-192) of Li Renwei has discussed the content determination of cryptotanshinone and tanshinone IIA by different processing methods, the influence of different addition methods on salvianolic acid B and alcohol-soluble extract is discussed in a paper (modern Chinese medicine), the two methods only adopt the high performance liquid chromatography to detect the content of the effective components, and can not scientifically reflect the quality of the salvia miltiorrhiza decoction pieces, because the traditional Chinese medicine plays a comprehensive medical role by depending on various contained chemical components, the effectiveness and specificity of the traditional Chinese medicine quality evaluation method based on the qualitative and quantitative properties of a certain chemical component are gradually questioned. Therefore, the inventor carries out fingerprint spectrum research on salvia miltiorrhiza, and the fingerprint spectrum does not emphasize the absolute uniqueness (individual specificity) of an individual but emphasizes the similarity of the same medicinal material group, namely the uniqueness (common characteristic) in the medicinal material group. The difference from the traditional quality control mode is that the fingerprint is comprehensively viewed, namely the 'complete face' of the chemical map, namely the integrity is emphasized, and the reflected quality information is comprehensive. Fingerprint analysis emphasizes accurate identification rather than precise calculation, and comparison maps emphasize similarity rather than identity. Under the condition that complex ingredients of the traditional Chinese medicine cannot be clarified, the fingerprint spectrum has the effect of reflecting the uniformity and stability of the internal quality of the traditional Chinese medicine.

In the prior art, HPLC fingerprint researches on salvia miltiorrhiza decoction pieces are established by madder and the like (journal of pharmacy in northwest, volume 28, 2013, 11 month, volume 28, stage 6), and the detection of the salvia miltiorrhiza decoction pieces by adopting an HPLC fingerprint is discussed in documents.

In order to solve the problems, the invention establishes the salvia miltiorrhiza medicinal material HPLC fingerprint spectrum measuring method aiming at different processing methods, the method is simple and convenient to operate, stable, high in precision and good in reproducibility, the common mode of the established salvia miltiorrhiza medicinal material HPLC fingerprint spectrum is shown in the analysis result of the correlation coefficient, the similarity of the salvia miltiorrhiza medicinal material and the salvia miltiorrhiza medicinal material of different processing methods is mostly more than 0.900, the chemical components of the salvia miltiorrhiza medicinal material and the salvia miltiorrhiza medicinal material of different processing methods are relatively stable, and the safety and the effectiveness of the medicinal materials are improved.

Disclosure of Invention

The invention aims to overcome the technical problems in the background technology and provides a quality detection method for salvia miltiorrhiza medicinal materials.

The quality detection method of the salvia miltiorrhiza medicinal material is used for detecting products prepared by a direct drying processing method, a drying processing method after water washing, a fresh cutting method or a sweating method, and particularly adopts an HPLC fingerprint spectrum method to detect the salvia miltiorrhiza medicinal material, and comprises the steps of setting chromatographic conditions, preparing a test solution and preparing a reference solution.

The direct drying processing method comprises the following steps: taking fresh salvia miltiorrhiza, shaking out soil, removing reed heads and fibrous roots, and drying in the sun or in the shade or drying at 40-100 ℃.

The processing method for drying after water washing comprises the following steps: washing fresh salvia miltiorrhiza, removing reed heads and fibrous roots, and drying in the sun or in the shade or at 40-100 ℃.

The fresh-cutting method comprises the following steps: taking a fresh salvia miltiorrhiza medicinal material, shaking off soil or washing the fresh salvia miltiorrhiza medicinal material with the soil, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying in the shade or in the sun or drying at 40-100 ℃.

The sweating method comprises a sweating method after drying and a sweating method after drying;

taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, removing reed heads and fibrous roots, airing until weight is reduced to 1/3, piling up the medicinal material for sweating until the inner core of the root strip is changed from white to purple brown, and drying the sweating medicinal material in the shade or in the sun or drying the sweating medicinal material in a blast drying oven at 40-100 ℃;

the method for sweating after drying specifically comprises the following steps: taking fresh salvia miltiorrhiza medicinal materials, shaking off soil, removing reed heads and fibrous roots, placing the medicinal materials in a blast drying oven, drying at 40-100 ℃ until weight is reduced to 1/3, piling the medicinal materials to perform sweating until the inner core of the root strip is changed from white to purple brown, and drying at 60 ℃ after the sweating is finished.

The method for detecting the salvia miltiorrhiza medicinal material by adopting the HPLC fingerprint spectrum method specifically comprises the following steps:

chromatographic conditions are as follows: column wondasil c18-WR, specification: 4.6mm multiplied by 250mm, 5 mu m, 1 percent glacial acetic acid water-methanol is taken as a mobile phase, the mobile phase elution is carried out according to a specified elution program, the detection wavelength is 210 nm-320 nm, the flow rate is 1mL min-1, the column temperature is 25-35 ℃, the sample injection amount is 10 mu L, the elution time is 60min, and the mobile phase elution program is as follows:

preparation of a test solution: precisely weighing 2.0g of salvia miltiorrhiza medicinal powder, placing the powder into a 50mL conical flask with a plug, adding 25mL of 30-100% methanol, weighing the weight, ultrasonically extracting for 15-60 min, cooling, complementing the loss weight with 30-100% methanol, and filtering with a 0.45-micron microporous filter membrane to obtain a test solution.

Preparation of control solutions: accurately weighing cryptotanshinone control, and preparing into 0.0467 μ g/μ L solution with methanol; accurately weighing tanshinone IIA reference substance, and preparing into 0.0289 μ g/μ L solution with methanol; the salvianolic acid B reference sample was weighed precisely and made up with 60% methanol to 0.2625. mu.g/. mu.L.

Preferably, the method for detecting the salvia miltiorrhiza medicinal material by using the HPLC fingerprint spectrum method specifically comprises the following steps:

chromatographic conditions are as follows: column wondasil c18-WR, specification: 4.6mm multiplied by 250mm, 5 mu m, 1 percent glacial acetic acid water-methanol is taken as a mobile phase, the mobile phase elution is carried out according to a specified elution program, the detection wavelength is 254nm to 286nm, the flow rate is 1mL min < -1 >, the column temperature is 25 ℃ to 35 ℃, the sample injection amount is 10 mu L, the elution time is 60min, and the mobile phase elution program is as follows:

preparation of a test solution: precisely weighing 2.0g of salvia miltiorrhiza medicinal powder, placing the powder into a 50mL conical flask with a plug, adding 25mL of 60-90% methanol, weighing the weight, ultrasonically extracting for 30-45 min, cooling, complementing the loss weight with 60-90% methanol, and filtering with a 0.45-micrometer microporous filter membrane to obtain a test solution.

Further preferably, the method for detecting the salvia miltiorrhiza medicinal material by using the HPLC fingerprint spectrum method specifically comprises the following steps:

preparation of a test solution: precisely weighing 2.0g of salvia miltiorrhiza medicinal powder, placing the powder into a 50mL conical flask with a plug, adding 25mL of 70-90% methanol, weighing the weight, ultrasonically extracting for 30-40 min, cooling, complementing the loss weight with 70-90% methanol, and filtering with a 0.45-micron microporous filter membrane to obtain a test solution.

the chromatographic conditions are as follows: the column was Wondasill C18-WR, specification: 4.6mm × 250mm, 5 μm, using 1% glacial acetic acid water and methanol as mobile phase, eluting the mobile phase according to the specification, wherein the detection wavelength is 254nm, the flow rate is 1 mL/min-1, the column temperature is 30 ℃, the sample injection amount is 10 μ L, the elution time is 60min, and the mobile phase elution program is as follows:

preparation of a test solution: precisely weighing 2.0g of radix Salviae Miltiorrhizae powder, placing in 50mL conical flask with plug, adding 25mL of 90% methanol, weighing, ultrasonically extracting for 30min, cooling, supplementing the weight loss with 90% methanol, and filtering with 0.45 μm microporous membrane to obtain test solution;

Problems of the prior art and advantages of the present invention

First, the problem of the prior art:

chenrubifen and the like establish HPLC fingerprint researches on salvia miltiorrhiza decoction pieces (northwest pharmaceutical journal,

volume

28, 6, 11 months in 2013), and the method cannot better control the quality of medicinal materials because of lower chromatographic peak separation degree and fewer common peaks in the fingerprints.

Secondly, the invention has the beneficial effects that:

1. the quality detection method disclosed by the invention is used for systematically researching the quality conditions of the decoction pieces processed in different producing areas of salvia miltiorrhiza in combination with the fingerprint spectrum, and the quality conditions of the decoction pieces processed in different processing methods are clarified through experiments.

2. Through the investigation of methodologies such as precision test, stability test, reproducibility test and the like, the RSD values of the relative peak area and the relative retention time of all common peaks in the precision test, the stability test and the reproducibility test are less than 3%, which shows that the salvia miltiorrhiza fingerprint spectrum measuring method established by the invention meets the technical requirements established by the traditional Chinese medicine fingerprint spectrum, and can be used for measuring the salvia miltiorrhiza fingerprint spectrum.

3. The method for measuring the HPLC fingerprint of the salvia miltiorrhiza medicinal material is simple and convenient to operate, stable, high in precision and good in reproducibility, a common mode of the HPLC fingerprint of the salvia miltiorrhiza medicinal material is established, and the similarity of the salvia miltiorrhiza medicinal material and the salvia miltiorrhiza medicinal materials of different processing methods is mostly more than 0.900 according to the analysis result of correlation coefficients, so that the chemical components of the salvia miltiorrhiza medicinal material and the salvia miltiorrhiza medicinal materials of the different processing methods are relatively stable.

4. When an HPLC fingerprint spectrum measuring method is established, different extraction solvents of methanol, 90% methanol, 60% methanol and 30% methanol are respectively compared; the extraction effects of different extraction methods such as reflux extraction, cold-soaking extraction and ultrasonic extraction of ethanol, 90% ethanol, 60% ethanol and 30% ethanol are definite, the overall effect of ultrasonic extraction by 90% methanol is good, the operation method is simple, the time is saved, the extraction is complete, the method is stable and good in reproducibility, the obtained spectrum information amount is large, the water-soluble component and the fat-soluble component are contained, the requirement that the fingerprint spectrum has integrity is met, the information contained in the salvia miltiorrhiza bunge is reflected as much as possible, and the method can be used for evaluating the comprehensive quality of the salvia miltiorrhiza bunge.

5. The time for HPLC fingerprint determination was examined and a 3 hour chromatogram was recorded when fingerprint elution time was selected. The result shows that after 60min, basically no chromatogram appears, and meanwhile, the difference of different time and collecting and processing time is considered to ensure that the characteristic peaks of all samples can be detected, and the selection of 60min as the detection time is definitely preferred.

6. In the selection of the detection wavelength, the HPLC chromatograms of all wavelengths scanned between 190 nm and 320nm are compared and analyzed, and the result shows that the absorption is larger at the detection wavelength of 254 nm. Therefore, it was clarified that 254nm is preferable as the detection wavelength.

7. When a mobile phase system is selected, gradient elution and isocratic tests are respectively carried out on the mobile phase systems with different proportions of methanol-water, 1% of glacial acetic acid methanol-1% of glacial acetic acid water and different volume fractions, and the results show that the 1% of glacial acetic acid water-methanol system has a good gradient elution effect, the detected chromatographic peak is relatively comprehensive, the peak area is large, the separation effect is good, after the elution proportions of the mobile phase at different times are adjusted, the retention time of each chromatographic peak is moderate, the base line is stable and not easy to drift, meanwhile, the separation degree of the chromatographic peak is improved, the tailing phenomenon of a chromatogram is well avoided, and the fingerprint can be well analyzed.

Description of the drawings:

FIG. 1 comparison of spectra obtained at different detection wavelengths

FIG. 2 chromatograms from different chromatographic columns

FIG. 3 chromatogram of three mobile phase systems on the separation of Salvia miltiorrhiza

FIG. 4 HPLC finger print of mixed reference substance and Saviae Miltiorrhizae radix sample

FIG. 5 chromatogram of three extraction methods

FIG. 6 different solvent extraction chromatograms

FIG. 7 different ultrasound time chromatograms

FIG. 812 HPLC fingerprint spectrum common mode of Saviae Miltiorrhizae radix

FIG. 912 batch HPLC fingerprint of Salvia miltiorrhiza Bunge

FIG. 10 HPLC fingerprint of Saviae Miltiorrhizae radix without water drying

FIG. 11 HPLC fingerprint of radix Salviae Miltiorrhizae sample after washing with water

FIG. 12 HPLC finger print of dried Saviae Miltiorrhizae radix sample without cutting in water

FIG. 13 HPLC fingerprint of dried Saviae Miltiorrhizae radix sample after washing with water and fresh cutting

FIG. 14 HPLC fingerprint of dried radix Salviae Miltiorrhizae sample after air-drying and sweating

FIG. 15 HPLC fingerprint of dried sweating Danshen root sample

FIG. 16 comparison of HPLC finger prints of Saviae Miltiorrhizae radix samples with 12 batches of Saviae Miltiorrhizae radix medicinal materials common mode

FIG. 17 control peak of Salvia miltiorrhiza sample by different processing methods

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 processing method of Salvia miltiorrhiza Bunge-producing area-direct drying processing

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibrous root, and sun drying.

Example 2 Salvia miltiorrhiza Bunge habitat processing method-direct drying processing

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibrous root, and drying in the shade.

Example 3 Salvia miltiorrhiza Bunge habitat processing method-direct drying processing

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibrous root, and oven drying at 40 deg.C.

Example 4 Salvia miltiorrhiza Bunge habitat processing method-direct drying processing

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibrous root, and oven drying at 60 deg.C.

Example 5 Salvia miltiorrhiza Bunge habitat processing method-direct drying processing

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibrous root, and oven drying at 80 deg.C.

Example 6 Salvia miltiorrhiza Bunge habitat processing method-direct drying processing

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibrous root, and oven drying at 100 deg.C.

Example 7 Salvia miltiorrhiza Bunge producing area processing method-drying after water rescue

Taking fresh salvia miltiorrhiza, shaking off soil, washing with water, removing reed heads and fibrous roots, and drying in the sun.

Example 8 processing method of Salvia miltiorrhiza Bunge in producing area-drying after water rescue

Taking fresh salvia miltiorrhiza, shaking off soil, washing with water, removing reed heads and fibrous roots, and drying in the shade.

Example 9 Salvia miltiorrhiza Bunge producing area processing method-drying after water rescue

Taking fresh salvia miltiorrhiza, shaking off soil, washing with water, removing reed heads and fibrous roots, and drying at 40 ℃.

Example 10 processing method of Salvia miltiorrhiza Bunge in producing area-drying after water rescue

Taking fresh salvia miltiorrhiza, shaking off soil, washing with water, removing reed heads and fibrous roots, and drying at 60 ℃.

Example 11 processing method of Salvia miltiorrhiza Bunge in producing area-drying after water rescue

Taking fresh salvia miltiorrhiza, shaking off soil, washing with water, removing reed heads and fibrous roots, and drying at 80 ℃.

Example 12 processing method of Salvia miltiorrhiza Bunge in producing area-drying after water rescue

Taking fresh salvia miltiorrhiza, shaking off soil, washing with water, removing reed heads and fibrous roots, and drying at 100 ℃.

Example 13 fresh-cut method

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying in the shade.

EXAMPLE 14 fresh-cutting method

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying in the sun.

EXAMPLE 15 fresh-cut method

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying at 40 ℃.

EXAMPLE 16 fresh-cutting method

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying at 60 ℃.

Example 17 fresh-cutting method

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying at 80 ℃.

EXAMPLE 18 fresh-cutting method

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying at 100 ℃.

Example 19 Water snatching followed by fresh-cutting

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, washing with water, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying in the shade.

Example 20 fresh-cut method after Water snatching

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, washing with water, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying in the sun.

Example 21 Water snatching followed by fresh-cutting

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, washing with water, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying at 40 ℃.

Example 22-fresh-cut after Water snatching

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, washing with water, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying at 60 ℃.

Example 23 Water snatching followed by fresh-cutting

Taking a fresh salvia miltiorrhiza medicinal material, shaking off soil, washing with water, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying at 80 ℃.

Example 24 Water snatching followed by fresh-cutting

Taking a fresh salvia miltiorrhiza medicinal material, shaking out soil or washing with water, removing reed heads and fibrous roots, cutting into 2-4 mm slices, drying in the shade, and drying at 100 ℃.

EXAMPLE 25 perspiration after drying

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibrous root, air drying to reduce weight of 1/3, piling, and sweating until the inner core of root strip changes from white to purple brown, and drying in shade.

Example 26 perspiration after drying

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibril, air drying to reduce weight of 1/3, piling, and sweating until the inner core of the root strip changes from white to purple brown, and air drying the sweating medicinal material.

Example 27 perspiration after drying

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibril, air drying to reduce weight of 1/3, piling up, and sweating until the inner core of the root strip changes from white to purple brown, and oven drying at 40 deg.C in a blast drying oven.

Example 28 perspiration after drying

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibril, air drying to reduce weight of 1/3, piling up, and sweating until the inner core of the root strip changes from white to purple brown, and drying the sweating medicinal material in a blast drying oven at 60 deg.C.

Example 29 perspiration after drying

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibril, air drying to reduce weight of 1/3, piling up, and sweating until the inner core of the root strip changes from white to purple brown, and drying the sweating medicinal material in a forced air drying oven at 80 deg.C.

Example 30 perspiration after drying

Taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibril, air drying to reduce weight of 1/3, piling up, and sweating until the inner core of the root strip changes from white to purple brown, and oven drying at 100 deg.C in a blast drying oven.

Example 31 perspiration after baking

Taking fresh salvia miltiorrhiza, shaking off soil, removing reed heads and fibrous roots, placing in a blast drying oven, drying at 40 ℃ until weight is reduced to 1/3, piling up, and sweating until the inner core of the root strip is changed from white to purple brown, and drying at 60 ℃ after sweating is finished.

Example 32 perspiration after baking

Taking fresh salvia miltiorrhiza, shaking off soil, removing reed heads and fibrous roots, placing the red sage roots in a blast drying oven, drying at 60 ℃ until weight reduction of 1/3 is achieved, piling the red sage roots, performing stuffiness and sweating until the inner core of the root strips is changed from white to purple brown, and drying at 60 ℃ after the sweating is completed.

Example 33 perspiration after baking

Taking fresh salvia miltiorrhiza, shaking off soil, removing reed heads and fibrous roots, placing in a blast drying oven, drying at 80 ℃ until weight is reduced to 1/3, piling up, and sweating until the inner core of the root strip is changed from white to purple brown, and drying at 60 ℃ after sweating is finished.

Example 34 perspiration after baking

Taking fresh salvia miltiorrhiza, shaking off soil, removing reed heads and fibrous roots, placing in a blast drying oven, drying at 100 ℃ until weight is reduced to 1/3, piling up, and sweating until the inner core of the root strip is changed from white to purple brown, and drying at 60 ℃ after sweating is finished.

EXAMPLES 1 TO 34 the product was tested according to the test method of any one of examples 35 to 41

Example 35 detection method

The detection of the salvia miltiorrhiza medicinal material by the HPLC fingerprint spectrum method specifically comprises the following steps:

chromatographic conditions are as follows: column wondasil c18-WR, specification: 4.6mm × 250mm, 5 μm, using 1% glacial acetic acid water-methanol as mobile phase, eluting the mobile phase according to a specified elution program, wherein the detection wavelength is 210nm, the flow rate is 1 mL/min-1, the column temperature is 25 ℃, the sample injection amount is 10 μ L, the elution time is 60min, and the mobile phase elution program is as follows:

preparation of a test solution: precisely weighing 2.0g of radix Salviae Miltiorrhizae powder, placing in 50mL conical flask with plug, adding 25mL of 30% methanol, weighing, ultrasonically extracting for 15min, cooling, supplementing the weight loss with 30% methanol, and filtering with 0.45 μm microporous membrane to obtain test solution.

Example 36 detection method

chromatographic conditions are as follows: column wondasil c18-WR, specification: 4.6mm × 250mm, 5 μm, using 1% glacial acetic acid water-methanol as mobile phase, eluting the mobile phase according to a specified elution program, wherein the detection wavelength is 320nm, the flow rate is 1 mL/min-1, the column temperature is 35 ℃, the sample injection amount is 10 μ L, the elution time is 60min, and the mobile phase elution program is as follows:

preparation of a test solution: precisely weighing 2.0g of radix Salviae Miltiorrhizae powder, placing in 50mL conical flask with plug, adding 25mL of 100% methanol, weighing, ultrasonically extracting for 60min, cooling, supplementing the weight loss with 100% methanol, and filtering with 0.45 μm microporous membrane to obtain test solution.

Example 37 detection method

The method for detecting the salvia miltiorrhiza medicinal material by using the HPLC fingerprint spectrum method specifically comprises the following steps:

chromatographic conditions are as follows: column wondasil c18-WR, specification: 4.6mm × 250mm, 5 μm, using 1% glacial acetic acid water-methanol as mobile phase, eluting the mobile phase according to a specified elution program, wherein the detection wavelength is 254nm, the flow rate is 1 mL/min-1, the column temperature is 25 ℃, the sample injection amount is 10 μ L, the elution time is 60min, and the mobile phase elution program is as follows:

preparation of a test solution: precisely weighing 2.0g of radix Salviae Miltiorrhizae powder, placing in 50mL conical flask with plug, adding 25mL of 60% methanol, weighing, ultrasonically extracting for 30min, cooling, supplementing the weight loss with 60% methanol, and filtering with 0.45 μm microporous membrane to obtain test solution.

Example 38 detection method

chromatographic conditions are as follows: column wondasil c18-WR, specification: 4.6mm × 250mm, 5 μm, using 1% glacial acetic acid water-methanol as mobile phase, eluting the mobile phase according to a specified elution program, wherein the detection wavelength is 286nm, the flow rate is 1 mL/min-1, the column temperature is 35 ℃, the sample injection amount is 10 μ L, the elution time is 60min, and the mobile phase elution program is as follows:

preparation of a test solution: precisely weighing 2.0g of radix salviae miltiorrhizae medicinal material powder, placing the powder into a 50mL conical flask with a plug, adding 25mL of 90% methanol, weighing the weight, ultrasonically extracting for 45min, cooling, complementing the loss weight with 60% -90% methanol, and filtering with a 0.45 μm microporous filter membrane to obtain a test solution.

Example 39 detection method

chromatographic conditions are as follows: column wondasil c18-WR, specification: 4.6mm × 250mm, 5 μm, using 1% glacial acetic acid water-methanol as mobile phase, eluting the mobile phase according to a specified elution program, wherein the detection wavelength is 254nm, the flow rate is 1 mL/min-1, the column temperature is 30 ℃, the sample injection amount is 10 μ L, the elution time is 60min, and the mobile phase elution program is as follows:

preparation of a test solution: precisely weighing 2.0g of radix Salviae Miltiorrhizae powder, placing in 50mL conical flask with plug, adding 25mL of 70% methanol, weighing, ultrasonically extracting for 30min, cooling, supplementing the weight loss with 70% methanol, and filtering with 0.45 μm microporous membrane to obtain test solution.

Example 40 detection method

chromatographic conditions are as follows: column wondasil c18-WR, specification: 4.6mm × 250mm, 5 μm, using 1% glacial acetic acid water-methanol as mobile phase, eluting the mobile phase according to the specified elution program, with the detection wavelength of 286nm, the flow rate of 1 mL/min-1, the column temperature of 30 ℃, the sample injection amount of 10 μ L, the elution time of 60min, and the mobile phase elution program as follows:

preparation of a test solution: precisely weighing 2.0g of radix salviae miltiorrhizae medicinal material powder, placing the powder into a 50mL conical flask with a plug, adding 25mL of 90% methanol, weighing the weight, ultrasonically extracting for 40min, cooling, complementing the weight loss with 70% -90% methanol, and filtering with a 0.45 μm microporous filter membrane to obtain a test solution.

Example 41 detection method

In order to further verify the scientificity and feasibility of the invention, the inventor carries out a large number of experiments, which are as follows:

experimental example 1 study on HPLC fingerprint of Salvia miltiorrhiza Bunge medicinal materials by different processing methods

1 materials of the experiment

1.1 Experimental instruments

Shimadzu high performance liquid chromatograph (LC-20AT, Shimadzu, Japan); one thousandth electronic balance (JT5003, jinuo balance instruments ltd, yao city); one in ten thousand electronic balance (model AUW220D, shimadzu instruments ltd), ultrasonic cleaning machine (HS10260D, tianjin hou oslo technologies); vacuum drying oven (DZ-2BC, Tensted instruments, Tianjin); a circulating water type vacuum pump (SHZ-DIII, Shanghai Diyu instruments Co., Ltd.); liwei high-speed universal disintegrator (Beijing liwei Yongxing instruments Co., Ltd.); digital display constant temperature water bath (HH-2, Australian instruments Co., Ltd., Changzhou).

1.2 reagent

The Saviae Miltiorrhizae radix is dried root and rhizome of Salvia officinalis Bge (Salvia militaria Bge.) of Labiatae, and is mainly produced in Shandong, Henan, Hebei, Sichuan and Anhui provinces. Guizhou has a salvia miltiorrhiza planting base. The 12 batches of Salvia miltiorrhiza medicinal materials used in the experiment are respectively collected in 12 different areas of China and are identified as the dry roots of Salvia miltiorrhiza (Salvia miliiorrhiza Bge.) in the family of labiatae by professor Wei sublimation in crude drug teaching and research room of Guizhou university of traditional Chinese medicine. The 12 batches are sourced as shown in table 1.

Methanol (pure chromatogram, Tianjin, Kemiou chemical reagent Co., Ltd., content is more than or equal to 99.9%); the methanol used for extraction is analytically pure (Fuyu fine chemical Co., Tianjin, mass fraction is more than or equal to 99.5%): glacial acetic acid (analytical purity, Tianjin Bodi chemical Co., Ltd.); child-haha purified water (manufactured by Hangzhou child-haha limited, entitled Guiyang child-haha Changsheng beverage); salvianolic acid B control (batch No. 115939-25-8) was purchased from Dai Biotech, Inc., Guizhou; cryptotanshinone control substances (batch No. 110852-200806) and tanshinone IIA (batch No. 110766-200619) are purchased from China pharmaceutical and biological product institute.

TABLE 1 sources of Salvia miltiorrhiza samples

1.3 preparation of Salvia miltiorrhiza samples with different processing methods

1.3.1 direct drying processing method

Taking 30kg of fresh salvia miltiorrhiza, shaking off soil, removing reed heads and fibrous roots, averagely dividing into 6 parts, and respectively drying in the sun, drying in the shade, and drying at 40 ℃, 60 ℃, 80 ℃ and 100 ℃.

1.3.2 drying process after water washing

Taking 30kg of fresh salvia miltiorrhiza, washing with water, removing reed heads and fibrous roots, averagely dividing into 6 parts, and respectively drying in the sun, drying in the shade, and drying at 40 ℃, 60 ℃, 80 ℃ and 100 ℃.

1.3.3 fresh-cutting method

(1) Taking 30kg of fresh salvia miltiorrhiza medicinal material, shaking off soil, removing reed heads and fibrous roots, cutting into 2-4 mm slices, averagely dividing into 6 parts, respectively drying in the shade, sun-drying, and drying at 40 ℃, 60 ℃, 80 ℃ and 100 ℃.

(2) Taking 30kg of fresh salvia miltiorrhiza, washing with water, removing reed heads and fibrous roots, cutting into 2-4 mm slices, averagely dividing into 6 parts, respectively drying in the shade, sun-drying, and drying at 40 ℃, 60 ℃, 80 ℃ and 100 ℃.

1.3.4 diaphoresis

(1) Sweating after drying: taking 30kg of fresh salvia miltiorrhiza medicinal material, shaking off soil, removing reed heads and fibrous roots, airing until weight is reduced to 1/3, piling up the medicinal material to perform sweating until the inner core of the root strip is changed from white to purple brown, averagely dividing the sweating medicinal material into 6 parts, respectively performing shade drying and sun drying, and drying in a blast drying oven at 40 ℃, 60 ℃, 80 ℃ and 100 ℃.

(2) Sweating after baking: taking 20kg of fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibril, dividing into 6 parts on average, respectively placing into blast drying oven, drying at 40 deg.C, 60 deg.C, 80 deg.C and 100 deg.C to reduce weight of 1/3 for sweating, and drying at 60 deg.C after sweating.

1.4 evaluation software

Chinese medicine chromatogram fingerprint similarity evaluation software system 2004 version A (national pharmacopoeia committee).

2 methods and results

2.1 selection of chromatographic conditions

2.1.1 selection of detection wavelength

The detection wavelength reported in the literature is often used for measuring the content of a specific component, and cannot meet the requirement of fingerprint spectrum research. The sample is scanned through the full wavelength of the ultraviolet visible spectrophotometer in the experiment, so that the absorption wavelength with larger absorption can be detected, and the wavelength can be conveniently selected. Four wavelengths, 210mm, 254mm, 286mm and 320mm, were selected for the assay in the experiment in combination with literature and full wavelength scanning. See fig. 1.

As can be seen from fig. 1: (1) under the detection wavelength of 210nm, the solvent peak is obvious, the absorption peak is few, and the baseline drift is obvious. (2) Under the detection wavelengths of 254nm and 286nm, the absorption peaks of the two are more, and the larger the information quantity is expressed. But at the detection wavelength of 286nm, part of the peak shape is wider. (3) And at the detection wavelength of 320nm, the absorption peak is less and the peak shape is wide.

And (4) conclusion: 254nm is selected as the detection wavelength of the HPLC fingerprint spectrum of the salvia miltiorrhiza.

2.1.2 selection of chromatography columns

Chromatographic columns of different brands and specifications are different in terms of carrier materials, silica gel purity, particle size, pore size, bonding phase type, density, bonding mode, end capping groups and technology, chromatographic column size, application range and the like. These differences are the main factors affecting the degree of separation of the components in the sample. The experiment examined the following columns of different manufacturers and different models (see table 2). And comparing the difference of the fingerprint spectra among different columns.

TABLE 2 different chromatography columns

Taking the same batch of sample (Sichuan Zhongjiang) liquid, and sequentially adopting different chromatographic columns to examine the separation effect of the sample on the salvia miltiorrhiza components. The results are shown in FIG. 2.

As can be seen from fig. 2, different chromatographic columns have a great influence on the fingerprint. Most of chromatographic peaks of the Welchrom C18 column basically reach baseline separation, but the chromatographic peaks are fewer than those of the Wondasil C18-WR column; analytical C18 column showed fewer peaks and no baseline separation was achieved; compared with Welchrom C18 column and Analytical C18 column, the Wondasil C18-WR column has more chromatographic peaks and the chromatographic peaks are basically separated from a base line. Through comparison of experimental results, the WndaSil C18-WR column is finally determined to be the experimental chromatographic column.

2.1.3 selection of the Mobile phase

After examining the relevant documents, the following three mobile phase systems were selected for experimental comparison, the mobile phase systems are shown in table 3, and the HPLC chromatogram is shown in fig. 3.

TABLE 3 different mobile phase systems

As can be seen from fig. 3: (1) performing gradient elution with methanol-water solution to obtain a spectrogram, wherein the baseline is shifted downwards, and chromatographic peaks are fewer than those of other mobile phase systems; (2) the chromatogram obtained by gradient elution of two mobile phase systems of 1% glacial acetic acid water-1% glacial acetic acid methanol and 1% glacial acetic acid water-methanol has more chromatographic peaks and better separation effect, and most peaks reach baseline separation. The former has a larger baseline drift after 40min than the latter. Due to the influence of factors such as instruments and sample introduction, the phenomenon that the base line fluctuates up and down in the first 2min of the chromatogram of the latter occurs, but the phenomenon has little influence on condition investigation. Therefore, the mobile phase was finally determined to be a 1% glacial acetic acid water-methanol system.

2.1.4 selection of mobile phase gradient elution procedure

Experiment the final elution procedure determined by adjusting the ratio of 1% glacial acetic acid in water and methanol is shown in table 4. The experimental process shows that: the main chromatographic peak of the sample achieves better separation in the elution time, and no chromatographic peak appears when the methanol proportion is more than 90 percent, so the highest methanol proportion is 90 percent, and the elution time is 60 min.

Table 4 mobile phase elution procedure

2.1.5 selection of column temperature

The investigation of 25 deg.C, 30 deg.C and 35 deg.C respectively shows that the column temperature has little influence on the peak pattern, and the column temperature of 30 deg.C is selected for determination.

2.1.6 optimum condition for fingerprint spectrum determination of red sage root

From the above experimental results, the final chromatographic conditions were determined to be: the column was Wondasill C18-WR (4.6 mm. times.250 mm, 5 μm), and the mobile phase elution program was as shown in Table 4, with a detection wavelength of 254nm, a flow rate of 1 mL. min-1, a column temperature of 30 ℃, a sample volume of 10 μ L, and an elution time of 60 min. Under the condition, the HPLC chromatogram of the mixed control solution and the sample of the radix Salviae Miltiorrhizae medicinal material is shown in FIG. 4.

2.2 selection of conditions for preparation of test article

2.2.1 examination of extraction methods

Methodology studies were performed according to the extraction methods referred to in the literature. Three extraction methods, namely a reflux method, an ultrasonic method and a cold soaking method, are respectively examined. Precisely weighing 3 parts of the powder of the salvia miltiorrhiza in the same batch by about 2.0g, respectively placing the powder in 50mL conical bottles with stoppers, precisely adding 25mL of 100% methanol by a pipette, weighing the weight, respectively extracting by the three methods for 30min, cooling, weighing the weight, complementing the lost weight with methanol, filtering, and taking the subsequent filtrate for later use. Pipetting 10. mu.L was measured using chromatographic conditions under item 2.1.1. The results are shown in FIG. 5.

From the whole chromatogram analysis (see fig. 5), the cold soaking and ultrasonic extraction solution has more chromatographic peaks and larger absorption in the chromatogram, and the reflux method has solvent peaks and tailing peaks. And (4) determining an ultrasonic extraction method as the extraction method of the experiment by combining the literature.

2.2.2 screening of extraction solvent

After looking up relevant literature data, methanol, 90% methanol, 60% methanol and 30% methanol are selected in sequence in the experiment; ethanol, 90% ethanol, 60% ethanol, 30% ethanol as solvent for examination. Precisely weighing 8 parts of the powder of the same batch of salvia miltiorrhiza medicinal materials by about 2.0g, respectively placing the powder into 50mL conical flasks with stoppers, precisely adding 25mL of each solvent by a pipette, weighing the weight, ultrasonically extracting for 30min, cooling, weighing the weight, complementing the lost weight by the corresponding solvent, filtering, and taking the subsequent filtrate for later use. Pipetting 10. mu.L and measuring according to the chromatographic conditions under item 2.1.1 gave the results shown in FIG. 6.

Global analysis from chromatogram (see fig. 6): the extraction effects of the methanol and the 90% methanol are basically similar, and the chromatographic peaks are more; because the peak area of the 90% methanol spectrum is slightly larger than that of the methanol spectrum, and because the 90% methanol is more economical than the methanol, the extraction solvent of the experiment is finally determined to be 90% methanol.

2.2.3 selection of extraction time

Through single factor investigation of 2.1.2.1 items and 2.1.2.2 items, the ultrasonic extraction method and the solvent are determined, and the extraction time is selected on the basis. Ultrasonic extraction with 90% methanol as solvent, and examining four extraction times of 15min, 30min, 45min and 60min respectively.

From the overall analysis of the chromatogram (see fig. 7), the 45min chromatogram has obvious solvent peaks and fewer chromatographic peaks; the chromatogram of 15min, 30min and 60min are similar, and screening should be performed between 15min and 30min to save time, and the chromatographic peak area of the 30min chromatogram is slightly larger than that of 15 min. The extraction time was finally determined to be 30 min.

2.2.4 determination of extraction conditions

Taking 100g of salvia miltiorrhiza medicinal material, crushing, sieving by a 40-mesh sieve, taking about 2.0g of sample powder, precisely weighing, placing in a conical flask with a plug, adding 25mL of 90% methanol, weighing, carrying out ultrasonic extraction for 30min, cooling, and complementing 90% of weight loss. Filtering, and filtering with 0.45 μm microporous membrane to obtain the final product.

2.2.5 preparation of test solutions

Precisely weighing 2.0g of Saviae Miltiorrhizae radix powder, placing in 50mL conical flask with plug, adding 25mL of methanol, weighing, ultrasonically extracting for 30min, cooling, supplementing the weight loss with methanol, and filtering with 0.45 μm microporous membrane to obtain test solution.

2.2.6 preparation of control solutions

Accurately weighing cryptotanshinone control, and preparing into 0.0467 μ g/μ L solution with methanol; accurately weighing tanshinone IIA reference substance, and preparing into 0.0289 μ g/μ L solution with methanol; the salvianolic acid B reference sample was weighed precisely and made up with 60% methanol to 0.2625. mu.g/. mu.L.

2.2 methodological investigation

2.2.1 precision test

Taking No. 1 (Sichuan Zhongjiang) Salvia miltiorrhiza Bunge medicinal powder to prepare a sample solution according to item 2.2.6, continuously feeding samples for 6 times under the chromatographic conditions, recording the fingerprint, and obtaining the RSD values of the relative retention time and the relative peak area of each main chromatographic peak less than 3% according to the measurement results shown in tables 5 and 6, wherein the RSD values meet the requirement of the fingerprint. Indicating that the instrument is accurate.

TABLE 5 relative peak area of main chromatographic peak for precision test

TABLE 6 precision test relative retention time of main chromatographic peaks

2.2.2 stability test

Taking No. 1 (Sichuan Zhongjiang) Salvia miltiorrhiza powder to prepare a sample solution according to item 2.3, determining fingerprint spectra according to the chromatographic conditions at 0, 2, 4, 8, 12 and 24h respectively, wherein the determination results are shown in tables 7 and 8, and the RSD values of the relative retention time and the relative peak area of each main chromatographic peak are less than 3%, which can meet the requirement of the fingerprint spectra and indicate that the components of the sample solution are stable within 24 h.

2.2.3 repeatability test

Taking 6 parts of No. 1 Salvia miltiorrhiza powder, preparing a parallel solution of a sample according to the method under item 2.3, and respectively injecting samples under the chromatographic conditions and recording fingerprint. The determination results are shown in tables 9 and 10, the RSD value of each main chromatographic peak relative retention time and peak area is less than 3%, and the method can meet the requirement of a fingerprint spectrum, thereby indicating that the method has good repeatability.

TABLE 7 stability test relative peak area of main chromatographic peak

Table 8 stability test main chromatographic peak relative retention time

TABLE 9 repeatability test relative peak area of main chromatographic peak

TABLE 10 repeatability tests relative retention time of the main chromatographic peaks

2.3 establishment of HPLC fingerprint of Salvia miltiorrhiza Bunge and technical parameters

2.3.1 comparison and analysis of HPLC finger print of Salvia miltiorrhiza Bunge

Using 12 batches of salvia miltiorrhiza collected in 12 different areas of China under 1.2 items as materials, preparing a test solution according to a method under 2.1.2.6 items, respectively sucking 10 mu L of sample injection, recording HPLC fingerprints, and then introducing the fingerprints of the 12 batches of medicinal materials into a Chinese medicine chromatography fingerprint similarity evaluation software system 2004A (national pharmacopoeia committee). Setting a matching template, automatically matching chromatographic peaks, generating a comparison fingerprint spectrum, and calculating the similarity of 1-12 batches of samples by taking a common mode as a reference spectrum. From the similarity data table 11 and fig. 9, it can be seen that the similarity of the salvia miltiorrhiza medicinal material in each batch is between 0.964 and 0.999, and the similarity is more than 0.900, which meets the specifications of technical requirements (temporary) for the traditional Chinese medicine injection fingerprint spectrum research.

Comparing the detection results of the chromatograms of different test samples, it is concluded that 15 common peaks in the fingerprint are shown in fig. 8, and comparing with the reference sample respectively confirms that the peak (5) is salvianolic acid B, the peak (11) is cryptotanshinone, and the peak (15) is tanshinone IIA.

Table 1112 batch Salvia miltiorrhiza Bunge medicinal materials fingerprint similarity analysis results

2.3.2 calibration of reference Peak of Salvia miltiorrhiza

By comparing the measurement results of the fingerprint spectra of 12 salvia miltiorrhiza medicinal materials with different producing areas, 15 chromatographic peaks are determined as a common peak, and a reference peak of salvianolic acid B No. 5 is selected (the separation degree of the salvianolic acid B from the adjacent chromatographic peaks in the fingerprint spectra of the salvia miltiorrhiza medicinal materials is more than 1.0, the retention time is proper, the peak area integral value is large, the intensity is large, the peak shape is stable, and in addition, the salvianolic acid B is one of the main effective components of the salvia miltiorrhiza medicinal materials, and a reference product can be purchased, so the salvianolic acid B is selected as a reference substance). The relative retention time and the relative peak area of the common peak in each fingerprint are counted, and the calculation results are shown in tables 12 and 13.

Relative peak area of HPLC fingerprint common peak of Saviae Miltiorrhizae radix in Table 1212 batches

TABLE 1312 batch of Salvia miltiorrhiza Bunge HPLC fingerprint spectrum common peak relative retention time

The result shows that the relative peak areas and relative retention times of the salvia miltiorrhiza medicinal material fingerprints of 12 different batches are basically consistent, the total peak accounts for 90.16 of the total peak area, and the established salvia miltiorrhiza HPLC fingerprint determination method meets the technical requirements established by the traditional Chinese medicine fingerprints and can be used for determining the salvia miltiorrhiza fingerprints.

2.4 comparison of HPLC finger prints of Saviae Miltiorrhizae radix by different processing methods

2.4.1 comparison of HPLC finger prints of radix Salviae Miltiorrhizae processed without water rescue

Introducing the HPLC fingerprint measured by the salvia miltiorrhiza sample which is not subjected to water-robbing drying into traditional Chinese medicine chromatogram fingerprint similarity evaluation system software (published by the national pharmacopoeia committee, 2004 edition), setting a matching template thereof, automatically matching chromatographic peaks, generating a comparison fingerprint, superposing the fingerprints as shown in figure 10, and performing similarity evaluation on the fingerprint of the salvia miltiorrhiza sample which is not subjected to water-robbing drying, wherein the result is shown in table 14. The similarity of the fingerprint spectrums of the salvia miltiorrhiza samples which are not subjected to water-robbing drying is more than 0.994, and the similarity is good, which indicates that the salvia miltiorrhiza medicinal material which is not subjected to water-robbing drying has high consistency of the whole chemical components.

TABLE 14 evaluation of HPLC fingerprint similarity of medicinal materials of salvia miltiorrhiza

2.4.2 comparison of HPLC finger prints of dried Saviae Miltiorrhizae radix after water washing

The similarity evaluation method of fingerprint chromatogram determined by water-washed and dried Saviae Miltiorrhizae radix sample is the same as item 2.3.1, the overlay figure is shown in figure 11, and the similarity result is shown in table 15. The table shows that the similarity of the fingerprint spectrums of the salvia miltiorrhiza samples dried after being washed by water is more than 0.996, the similarity is good, and the high consistency of the whole chemical components of the salvia miltiorrhiza medicinal materials dried after being washed by water is proved.

TABLE 15 evaluation of HPLC fingerprint similarity of dried Salvia miltiorrhiza Bunge after washing with water

2.4.3 comparison of HPLC finger prints of fresh-cut Salvia miltiorrhiza Bunge

The similarity evaluation method for fingerprint spectrum of fresh-cut and dried Saviae Miltiorrhizae radix sample without robbing water is 2.3.1, the superimposed graph is shown in FIG. 12, and the similarity result is shown in Table 16. The table shows that the similarity of the fingerprint spectrums of the salvia miltiorrhiza samples which are not subjected to water robbing and fresh cutting and drying is more than 0.981, the similarity is good, and the high consistency of the whole chemical components of the salvia miltiorrhiza medicinal materials which are not subjected to water robbing and fresh cutting and drying is proved.

TABLE 16 evaluation of HPLC fingerprint similarity of dried Saviae Miltiorrhizae radix after fresh-cut without robbing water

2.4.4 comparison of HPLC finger prints of fresh-cut Salvia miltiorrhiza Bunge after washing with water

The similarity evaluation method of fingerprint measured on fresh-cut and dried Saviae Miltiorrhizae radix sample after water washing is the same as item 2.3.1, the superimposed graph is shown in FIG. 13, and the similarity result is shown in Table 17. The table shows that the fingerprint similarity of the fresh-cut and dried salvia miltiorrhiza samples after water washing is over 0.970, the similarity is good, and the high consistency of the whole chemical components of the fresh-cut and dried salvia miltiorrhiza medicinal materials after water washing is proved.

TABLE 17 evaluation of HPLC fingerprint similarity of fresh-cut dried Salvia miltiorrhiza Bunge after washing with water

2.4.5 comparison of HPLC finger prints of radix Salviae Miltiorrhizae by sweating after air drying

The similarity evaluation method of fingerprint obtained by sweating method after air drying is the same as item 2.3.1, the superimposed graph is shown in FIG. 14, and the similarity result is shown in Table 18. The table shows that the similarity of the fingerprint spectrum of the salvia miltiorrhiza sample obtained by the sweating method after drying is over 0.960, the similarity is good, and the high consistency of the whole chemical components of the salvia miltiorrhiza medicinal material which is fresh cut and dried after washing is proved.

TABLE 18 evaluation of HPLC fingerprint similarity of dried Saviae Miltiorrhizae radix after drying to reduce weight 1/3 and sweating

2.4.6 comparison of HPLC finger prints of dried and sweated Saviae Miltiorrhizae radix

Similarity evaluation method for fingerprint obtained by drying and sweating Saviae Miltiorrhizae radix sample is the same as item 2.3.1, the superimposed graph is shown in FIG. 15, and the similarity result is shown in Table 19. It can be seen from the table that the similarity of the fingerprint spectrum of the salvia miltiorrhiza sample obtained by the drying sweating method is more than 0.900 except that the sample S4 is 0.822<0.900, which indicates that the uniformity of the whole chemical components of the salvia miltiorrhiza medicinal material obtained by the drying sweating method is not good compared with the 5 processing methods.

TABLE 19 evaluation of HPLC fingerprint similarity of Saviae Miltiorrhizae radix material dried at different temperatures to reduce weight, 1/3, induce sweating, and dried at 60 deg.C

2.4.7 comparison of common mode of radix Salviae Miltiorrhizae with 12 batches of radix Salviae Miltiorrhizae

On the basis of establishing a relatively stable salvia miltiorrhiza medicinal material fingerprint analysis and test method, fingerprint identification is carried out on salvia miltiorrhiza samples obtained by different processing methods. Weighing 34 parts of Saviae Miltiorrhizae radix powder obtained by drying to constant weight by different processing methods, sieving with 40 mesh sieve, preparing test solution according to the method of '2.1.2.6 items' for each part, determining according to the chromatographic conditions of '2.1.1.6 items', and recording its HPLC chromatogram. And (3) performing similarity analysis on the salvia miltiorrhiza sample fingerprints obtained by different processing methods by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2004 edition). The similarity is represented by median, and compared with the common pattern of the reference finger print of 12 batches of salvia miltiorrhiza medicinal materials, the result is shown in table 20 and fig. 16.

It can be known from the table that the fingerprint of the salvia miltiorrhiza bunge medicinal material obtained by different processing methods has the common mode similarity with the salvia miltiorrhiza bunge medicinal material of 12 batches, and the samples are all over 0.800 except that the differences of the samples S31, S32 and S33 are less than 0.800. 15 common peaks in chromatogram obtained by HPLC fingerprint peak common mode of 12 batches of salvia miltiorrhiza medicinal materials with different producing areas can be used as fingerprint characteristics for identifying the quality of salvia miltiorrhiza medicinal materials with different producing areas and different processing methods.

TABLE 20 results of comparing similarity between common mode of Saviae Miltiorrhizae radix and 12 batches of Saviae Miltiorrhizae radix

2.4.8 comparative study of main characteristic peak area of Saviae Miltiorrhizae radix by different processing methods

Comparing the detection results of the chromatograms of different test samples, 17 common peaks in the fingerprint are summarized in tables 21, 22 and 23 and fig. 17 (the sequence of the sample numbers and names is the same as table 20), and comparing with the reference sample, the peak (4) is confirmed to be salvianolic acid B, the peak (13) is cryptotanshinone, and the peak (17) is tanshinone IIA. It can be seen from the figures and tables that the five processing methods of the direct processing method, the water-robbing post-processing method, the direct fresh-cutting method, the water-robbing post-fresh-cutting method and the sweating method have the same number of common peaks and the same peak emergence time, but have different peak shapes and larger peak area differences. Comparing 34 salvia miltiorrhiza samples with different processing methods, the sample S17 (directly fresh-cut and dried in the sun) has 5 common peaks with the maximum peak areas of 7, 8, 9, 10 and 12, and the common peaks of the sample No. 4, 14 and 17 are close to the maximum peak areas; the peak areas of 5 common peaks of the S10 sample (washed and dried at 100 ℃) are the minimum, namely, the

peaks

6, 8, 15, 16 and 17, and the

peaks

7, 10, 11 and 13 of the sample are close to the minimum common peak area; the minimum relative peak areas of 4 common peaks of the sample S31 (dried at 40 ℃ and dried at 1/3 for sweating and 60 ℃) are respectively No. 1, No. 3, No. 4 and No. 5; the peak areas of 3 common peaks of the S11 sample (washed and dried in the sun) are respectively No. 12, No. 13 and No. 14.

Because the experimental extraction solvent is 90% methanol, the mobile phase is a glacial acetic acid water-methanol system, and the used chromatographic column is Wondasill C18-WR (4.6mm multiplied by 250mm, 5 μm) reverse chromatographic column, the water-soluble component with large polarity in the salvia miltiorrhiza firstly elutes out the peak, and the fat-soluble component with small polarity later elutes out the peak. Therefore, the sample No. S17 (directly cut and dried in the sun) is beneficial to the dissolution of the polar neutral components. The S10 sample (washed and dried at 100 ℃) is not suitable for dissolving neutral and polar small components. The sample S31 (dried at 40 ℃ to reduce 1/3 to induce sweating and dried at 60 ℃) is not suitable for the dissolution of acute major components. The S11 sample (washed and dried in the sun) is not suitable for dissolving out the polar small components.

TABLE 21 common Peak area of Salvia miltiorrhiza samples from different processing methods

TABLE 22 common Peak area of Salvia miltiorrhiza samples for different processing methods

TABLE 23 common Peak area of Salvia miltiorrhiza samples from different processing methods

3 conclusion and discussion

3.1 comparison of HPLC finger prints of Saviae Miltiorrhizae radix by different processing methods

From the results of HPLC fingerprint measurement of different processing methods under item 2.4, the influence of the processing method on the overall chemical composition of the salvia miltiorrhiza medicinal material is obvious, except that the similarity of the samples S25 (dried by air and sweated and then dried in the sun), S30 (dried by 100 ℃ after dried and sweated), S31 (dried by drying at 40 ℃ and then dried by drying at 1/3 and then dried at 60 ℃), S32 (dried by drying at 60 ℃ and then dried by drying at 1/3 and then dried by drying at 60 ℃) and S33 (dried by drying at 80 ℃ and then dried by drying at 1/3 and then dried at 60 ℃) in the sweating method are respectively lower, and the similarity of the salvia miltiorrhiza samples of other processing methods is more than 0.9, which shows that the influence on the overall chemical composition of the salvia miltiorrhiza medicinal material is smaller. Compared with other processing methods, the sweating method has the advantages that the whole peak area of the water-soluble characteristic peak (peak No. 4) is reduced, and particularly the sweating method after drying is greatly changed; the phenomenon that the whole peak areas of characteristic peaks (peak 13 and peak 17) of fat-soluble components are large and even increased is probably the reason of low similarity, and whether new chemical components and curative effects are generated in the process of sweating is to be further studied in depth by combining pharmacology.

3.2 selection and optimization of chromatographic conditions

In the selection of the detection wavelength, HPLC chromatograms of scanned wavelengths scanned between 190-320 nm are compared and analyzed, and as a result, the detection is carried out at 254nm, and the reference substance cinnamic acid has larger absorption at the wavelength. Therefore, it is taken as the detection wavelength; when a mobile phase system is selected, through examination of related documents, gradient elution and isocratic tests are respectively carried out on the mobile phase system with methanol-water, 1% glacial acetic acid methanol-1% glacial acetic acid water, methanol-1% glacial acetic acid water in different proportions and different volume fractions, and the results show that the 1% glacial acetic acid water-methanol system has a good gradient elution effect, the detected chromatographic peaks are relatively comprehensive, the peak area is large, the separation effect is good, after the elution proportions of the mobile phase are adjusted in different times, the retention time of each chromatographic peak is moderate, the base line is stable and not easy to drift, the separation degree of the chromatographic peaks is improved, the tailing phenomenon of the chromatogram is well avoided, and the fingerprint can be well analyzed.

3.3 selection of extraction conditions for sample solution and time for measuring finger print

The test compares different extraction solvents methanol, 90% methanol, 60% methanol, 30% methanol, respectively; the extraction effects of different extraction methods such as reflux extraction, cold-soaking extraction and ultrasonic extraction of ethanol, 90% ethanol, 60% ethanol and 30% ethanol are good, the result is that the overall effect of ultrasonic extraction with 90% methanol is good, the operation method is simple, the time is saved, the extraction is complete, and the method is stable and has good reproducibility. In addition, the obtained spectrum information amount is large, the obtained spectrum information amount contains water-soluble components and fat-soluble components, the requirement that the fingerprint spectrum has integrity is met, the information contained in the salvia miltiorrhiza medicinal material is reflected as much as possible, and the method can be used as a method for evaluating the comprehensive quality of the salvia miltiorrhiza medicinal material. In addition, the HPLC fingerprint measurement time was also examined and when fingerprint elution time was selected, a chromatogram of 3 hours was recorded. The result shows that after 60min, basically no chromatogram appears, and the difference of different time and collecting and processing time is considered to ensure that the characteristic peaks of all samples can be detected, and 60min is selected as the detection time.

3.4 examination of fingerprint detection methodology

Through the respective investigation of methodologies such as a precision test, a stability test, a reproducibility test and the like, the RSD values of the relative peak area and the relative retention time of all common peaks in the precision test, the stability test and the reproducibility test are less than 3%, which shows that the salvia miltiorrhiza fingerprint spectrum determination method established in the test meets the technical requirements established by the traditional Chinese medicine fingerprint spectrum, and can be used for determining the salvia miltiorrhiza fingerprint spectrum.

Traditional Chinese medicines play a comprehensive medical role by depending on various contained chemical components, and the effectiveness and specificity of a traditional Chinese medicine quality evaluation method based on the qualitative and quantitative properties of a certain chemical component are gradually questioned. Fingerprints do not emphasize absolute uniqueness (individual specificity) of an individual, but emphasize similarity of the same medicinal material group, i.e. uniqueness within the group of materials (common characteristics). The difference from the traditional quality control mode is that the fingerprint is comprehensively viewed, namely the 'complete face' of the chemical map, namely the integrity is emphasized, and the reflected quality information is comprehensive. Fingerprint analysis emphasizes accurate identification rather than precise calculation, and comparison maps emphasize similarity rather than identity. Under the condition that complex ingredients of the traditional Chinese medicine cannot be clarified, the fingerprint spectrum has the effect of reflecting the uniformity and stability of the internal quality of the traditional Chinese medicine.

The HPLC fingerprint determination method for the salvia miltiorrhiza medicinal material established in the experiment is simple and convenient to operate, stable, high in precision and good in reproducibility, a common mode of the HPLC fingerprint of the salvia miltiorrhiza medicinal material is established, and the similarity of the salvia miltiorrhiza medicinal material and the salvia miltiorrhiza medicinal materials of different processing methods is mostly more than 0.900 according to the analysis result of correlation coefficients, so that the chemical components of the salvia miltiorrhiza medicinal material and the salvia miltiorrhiza medicinal materials of the different processing methods are relatively stable.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A quality detection method for Saviae Miltiorrhizae radix is characterized in that the detection method is used for detecting products prepared by direct drying processing method, or drying processing method after water washing, or fresh cutting method or sweating method, and specifically adopts HPLC fingerprint chromatogram method to detect Saviae Miltiorrhizae radix, including setting chromatographic conditions, preparing test solution, and preparing reference solution.

2. The quality inspection method according to claim 1, wherein the direct drying process comprises: taking fresh salvia miltiorrhiza, shaking out soil, removing reed heads and fibrous roots, and drying in the sun or in the shade or drying at 40-100 ℃.

3. The quality detection method according to claim 1, wherein the processing method of drying after water-washing comprises: washing fresh salvia miltiorrhiza, removing reed heads and fibrous roots, and drying in the sun or in the shade or at 40-100 ℃.

4. The quality inspection method according to claim 1, wherein the fresh-cut method is: taking a fresh salvia miltiorrhiza medicinal material, shaking off soil or washing the fresh salvia miltiorrhiza medicinal material with the soil, removing reed heads and fibrous roots, cutting into 2-4 mm slices, and drying in the shade or in the sun or drying at 40-100 ℃.

5. The quality inspection method according to claim 1, wherein the sweating method includes a post-drying sweating method and a post-drying sweating method.

6. The quality detection method according to claim 5, wherein the air-dried sweating method comprises taking fresh Saviae Miltiorrhizae radix, shaking off soil, removing rhizoma Phragmitis and fibrous root, air-drying until weight is reduced to 1/3, piling up, and sweating until the inner core of root strip is changed from white to purple brown, drying or sun-drying the sweating medicinal material in the shade or at 40-100 deg.C or oven-drying in a blast drying oven;

the method for sweating after drying specifically comprises the following steps; taking fresh salvia miltiorrhiza medicinal materials, shaking off soil, removing reed heads and fibrous roots, placing the medicinal materials in a blast drying oven, drying at 40-100 ℃ until weight is reduced to 1/3, piling the medicinal materials to perform sweating until the inner core of the root strip is changed from white to purple brown, and drying at 60 ℃ after the sweating is finished.

7. The quality detection method according to claim 1, wherein the detection of the salvia miltiorrhiza medicinal material by the HPLC fingerprint spectrum method specifically comprises the following steps:

8. The quality detection method according to claim 7, wherein the detection of the salvia miltiorrhiza medicinal material by the HPLC fingerprint spectrum method specifically comprises the following steps:

9. The quality detection method according to claim 8, wherein the detection of the salvia miltiorrhiza medicinal material by the HPLC fingerprint spectrum method specifically comprises the following steps:

10. The quality detection method according to claim 9, wherein the detection of the salvia miltiorrhiza medicinal material by the HPLC fingerprint spectrum method specifically comprises the following steps: